Freeze tolerance is an important adaptation that promotes winter survival in several species of terrestrial vertebrates. In the wood frog (Rana sylvatica), which routinely survives the freezing of up to 65% of its body water, freeze tolerance is promoted by two primary adaptations. First, a reversible dehydration of organs during freezing, in which up to 60% of the tissue water is translocated to lymphatic and coelomic spaces, protects the microvasculature against mechanical cryoinjury. Secondly, glucose, which is rapidly mobilized during freezing from hepatic glycogen reserves, is the cryoprotectant that promotes cell and tissue survival. Within minutes of the onset of freezing, glucose concentrations in the liver and blood rapidly increase and ultimately may reach 150-300 mM. Although the biochemical processes regulating glycogenolysis within the hepatocytes are understood, the extra-hepatic mechanism triggering this response is as yet unknown. The present project will investigate the following hypotheses: I. The Cryoprotectant Mobilization Response is Under Sympathetic Control: First, we will determine whether the liver can auto-initiate glucose mobilization in direct response to freezing. Secondly, presuming that the regulatory mechanism is extra-hepatic, we will determine whether sympathetic reflexes at the level of the spinal cord or brain initiate cryoprotectant mobilization. Thirdly, we will critically evaluate the role of the sympathetic nervous system by demonstrating in vivo blockage of the glycogenolytic response to freezing in sympathectomized animals. II. Catecholamines Mediate the Cryoprotectant Mobilization Response: After characterizing hepatocyte adrenoceptors using Scatchard analyses, we will verify the efficacy of pharmacological and physiological agonists in stimulating glycogenolysis in cultured liver. Finally, we will critically evaluate the role of catecholamines in triggering glucose mobilization by demonstrating in vivo blockage of the glycogenolytic response to freezing in animals pre-treated with appropriate antagonists. Health Relatedness and Long-term Objectives: Currently it is not possible to cryopreserve human organs. Freeze-tolerant vertebrates, such as the wood frog, have "solved" not only the problem of successfully freezing individual organs, but that of simultaneously freezing entire organ systems. Studies of the mechanisms by which these animals survive extensive ice formation have already provided valuable clues for the development of successful methods for the cryopreservation and banking of human organs, as well as insight into the significant biomedical problems concerning glucose metabolism, ischemia and hypoxia, and electrolyte balance.